Microbial diseases have long been a major health concern worldwide. A key feature in the prevention of such diseases is early diagnosis. Epidemiologists must look for microbial contamination in the environment as well as in food products to find common causes of outbreaks.
One example is the outbreak in 1992 of Enterohemorrhagic E. coli (EHEC) in the Pacific Northwest of the United States due to contaminated ground beef. EHEC is a relatively "newly discovered" pathogen. EHEC was first isolated in 1975, and it was not until 1982 that E. coli 0157:H7 was associated with two food-related outbreaks of hemorrhagic colitis in the United States. The reported incidence of E. coli 0157:H7 cases is increasing. Typically, E. coli strains are harmless commensals, but a few strains are pathogenic. EHEC is particularly virulent and can trigger deadly complications, including severe abdominal cramps and acute renal failure in children as well as cardiovascular and central nervous system problems.
As another example, Salmonella is the leading cause (more than 50%) of total bacterial foodborne disease outbreaks, according to the United States Centers for Disease Control (CDC) surveillance of foodborne diseases. On average, there were 68 incidents and 6249 cases per year reported to the CDC during the period 1983-1987, the most recent summary period available. Salmonella can infect a broad variety of warm- and cold-blooded animals, and can survive for long periods of time outside a host.
Listeria, a genus of gram positive bacteria, is widely distributed in nature, having been isolated from soil, water, vegetation and many animal species. Serious outbreaks of human listeriosis have not been frequent, but have been identified with increasing incidence. In addition, the detection frequency for Listeria in the agricultural environment appears to be increasing. For specific outbreaks of listeriosis, estimates place mortality at 30% to 40% of affected patients, however, little is known of the minimum infective dose. One particularly troublesome aspect of Listeria control in foods is that Listeria can grow at temperatures as low as -0.4.degree. C. and as high as 44.degree. C. These factors all contribute to the increasing significance of Listeria as a food pathogen.
The ability to monitor potential environmental and food sources of microbial contamination quickly and easily would reduce the risk of human infection and potential mortality. A device able to assay for microorganisms, including bacteria, yeasts, molds, fungi, parasites and viruses, that requires no special or technical equipment, can be performed in the field and does not require special skills would be useful for diagnosis as well as environmental monitoring and food sampling. In the case of foodborne bacterial contamination, three of the major disease-related organisms are Salmonella, Listeria and EHEC.
There are a number of Salmonella, Listeria, and EHEC detection methods presently available. Trained laboratory technicians and a minimum of 2-5 days are required to obtain evidence of these organisms by the standard cultural methods of analysis. New, more rapid methods are based on such techniques as enzyme immunoassay (EIA), DNA hybridization, immunodiffusion, or growth/metabolism measurements. While taking much less time than the cultural methods, these rapid tests still require skilled technical training, a functional laboratory, and specialized equipment. These tests generally take two or more days total, including several hours of hands-on time. When looking at other developing technologies in the diagnostics field, such as flow cytometry and polymerase chain reaction (PCR), the instrumentation and technical skills that are required to accurately perform such tests render them inappropriate for use in food microbiology, environmental testing and physician's office diagnosis.
Another recent technology in the diagnostics field involves lateral flow sandwich immunoassays. Such tests have been developed for the detection of human chorionic gonadotropin (hCG), and applied to pregnancy testing. Typically, a monoclonal or polyclonal antibody is mobilized in a discrete band near the distal end of a solid carder strip, called the detection zone. Another mount of antibody is labeled with a detection reagent such as an inorganic sol or dyed polystyrene particle. This labeled antibody is reversibly fixed near the proximal end of the carrier strip. Upon hydration of the proximal end with a sample fluid potentially containing the antigen, the antigen reacts with the labeled antibody and the complex passes through the zone of mobilized antibody, forming a sandwich upon reacting with the immobilized antibody. The capture of the chromogenic reagent-antigen complex causes the formation of a visible signal in the detection zone.
There are at least two major challenges that must be addressed to distinguish pathogenic bacteria, as opposed to distinguishing hormones or other soluble molecular targets. These challenges are the need to detect all of the target strains of a target microorganism in the presence of numerous antigenically related organisms, with a low tolerance for false positive results and a very low, preferably zero, tolerance for false negatives, and the physical size and heterogeneity of the target itself. A typical clinical diagnostic test, such as a test for hCG in urine, is focused on detecting a single, small, unique entity (i.e., a hormone) in a well-characterized matrix (e.g., urine). Furthermore, the structure of the analyte (hCG), is defined and uniform in size and composition.
Pathogen detection, for example, a test for E. coli 0157:H7, must distinguish a particular pathogenic strain from nonpathogenic strains of the target microorganism. In contrast to the well-defined small size and structure of most hormones or marker proteins, microorganisms are very large and their surfaces are heterogeneous and can undergo changes, such as the phase-switching of Salmonella flagella.
In previous attempts to transfer the lateral flow technology of clinical chemistry to the detection of microorganisms, high affinity polyclonal antibodies were prepared against Salmonella, Listeria, and EHEC antigens. These antibodies were conjugated to chromogenic reagents such as dyed polystyrene particles and inorganic sols. Upon addition of the target microorganisms, rapid and pronounced agglutination occurred, resulting in large aggregates that prevented the flow of the chromogenic reagent-analyte complex along the solid carrier to the zone of capture antibody.
Thus, there is a need in the art for the adaptation of lateral flow technology for the detection of heterogeneous microorganisms in a variety of matrices. The present invention provides these and other, related advantages.